The current project addresses a novel regulatory mechanism of neuronal activity by microRNAs and its role in controlling animal behavior and epilepsy. We found that a neuron-enriched microRNA, miR-128, acts as a negative master modulator of neuronal signaling responses in mice. The importance of the miR-128 in brain function is underscored by the development of fatal epileptic seizures in mice that lack miR-128 in postnatal forebrain neurons. Accordingly, overexpression of miR-128 in neurons attenuates chemically-induced seizures and rescues survival in mice. These findings reveal miR-128 as a previously unknown key regulator of neuronal activity with a potential for the development of novel anti-epileptic therapeutic approaches. The major regulatory function of miR-128 highlights the importance of the mechanisms that control miR-128 expression levels as well as miR-128 access to its targets in neurons. We found that calcineurin/NFAT activity, an important signaling pathway in neuronal development and function, controls miR-128 expression in neurons. Most importantly, mice with neuronal calcineurin deficiency develop a fatal seizure syndrome similar to the one observed in mice with miR-128 deficiency. These findings provided a strong indication for the role of calcineurin/NFAT signaling in control of miR-128 expression. The mechanism of calcineurin/NFAT-mediated regulation of miR-128 expression will be addressed in this proposal. The proposal also addresses a novel mechanism of regulation of miR-128 effector function. Preliminary data suggest that access of miR-128 to its mRNA targets is regulated by miR-128-sequestring, non-coding decoy RNAs. This novel mechanism of regulation of miR-128 effector function will be explored in experiments using unique mouse models with inactivation or overexpression of the neuron-specific miR-128 decoy RNAs in combination with the molecular, electrophysiological and behavioral approaches that have been established in our laboratory. The central role of miR-128 in neuronal signaling makes this miRNA an attractive target for potential therapeutic intervention of epilepsy. Using a mouse model of the human Dravet syndrome, one the most severe and often treatment resistant forms of epilepsy, the proposal will explore the therapeutic potential of neuronal miR-128 modulation for the treatment of the disease. To bring the research closer to the development of actual antiepileptic therapy, we will explore the antiepileptic potential of exogenously expressed miR-128. MiR-128 will be delivered to defined neurons using neurotrophic adeno-associated viruses (AAV) that are currently considered as the most reliable vehicle for in vivo gene delivery in mice and human. In summary, our proposal describes highly innovative and hypothesis driven studies of a novel non-coding RNA mechanism for controlling neuronal signaling and activity that has a strong potential to be applied for the treatment of human epilepsy.